Abstract: The aim of the present study was to establish an approach to continuously record fluorescent signals of rat cerebral cortical neurons in vivo, using the novel system composed of fibre-optic probe and fluorescence microscopy. To visualize cortical neurons, recombinant virus vectors carrying green fluorescent protein (GFP) gene were microinjected into cerebral cortex in Sprague Dawley (SD) rats. Seven days later, an imaging microprobe, composed of optical minifibres, was inserted into the microinjected region of the cerebral cortex. By using the fibered fluorescence microscopy, we observed fluorescent signals of cortical neurons transfected with GFP in living animals. In the brain slices from the microinjected region, the fluorescence signals of GFP were recorded using fluorescence microscopy, which confirmed the observation of the fibered fluorescence microscopy. The novel technology established in the present study maintains the physical condition of the experimental animal, and meets the demands of fluorescence micro-imaging in neural tissue in vivo. Application of this technology allows a direct and rapid approach to tracing fluorescent signals of neurons in living animals.
Key words: in vivo molecular imaging technology; green fluorescent protein; neurons

With the development of modern molecular biology techniques, animals in in vivo experiments have become an indispensable part of neuroscience research, this including the rapid development in recent years of live animal molecular imaging techniques.

Neuroscience experimental protocol often requires microscopic observation of the experimental animal's neuronal structure or neuronal growth migration record and this requires microscopic imaging equipment to carry out observation externally and within quite a large scope of the animal's neurons or under minimally invasive conditions, thus carrying out real-time internal observation and recording of the animal's neurons. In previous studies, due to limited instruments and equipment as well as hardware configuration and imaging resolution limitations, it was not possible to carry out in vivo microscopic fluorescence imaging of the brain tissue's neurons with optimal results. Observation methods used on the internal tissue of animals, including PET, MRI [1], and multi-photon microscopy imaging techniques [2], all entail certain limitations. Although PET enables structural observation on a millimetric scale, it entails relatively slow speeds when capturing images and MRI image resolution and signal acquisition speeds are significantly lower than those of fluorescence microscopy. Although multiphoton microscopes, on the other hand, have higher imaging resolution rates and image capturing speeds, their observable tissue depth is limited to a depth of to 500 μm in animal skin, and it is therefore difficult to observe the structure of the cerebral cortex of mammals in much depth. With the increasing requirements in scientific research, the gradient index lens began to be applied to the study of the in vivo cerebral cortex [3,4], and this technology can be used with minimal invasiveness by penetrating the in vivo animal by a few millimetres with single or dual-colour fluorescence microscopy imaging, whilst however, in accordance with research requirements, still maintaining a certain distance from the in vivo neural activity during real-time observation. Since none of these techniques can fulfil the requirements of microscopic imaging for the in vivo animal target areas, many experiment protocols must limit their fluorescence microscopy observation of local animal tissue to in vitro tissue sections.
Recently, our technology platform introduced probe-type equipment for fluorescence microscopy imaging [5]. This equipment can, through a minimally invasive cut, incorporate 30 000 strands of flexible optic fibre probes placed inside the animal body's target area. Laser activation recording can then assess the area's fluorescent tag conditions and accomplish the intended purpose of the experimental observation. This paper aims to take advantage of the optic fibre in vivo fluorescence microscopy imaging system and establish the in vivo fluorescence microscopy continuous observation experiment methods for rat neurons.

1 Materials and methods
1.1 Animal Sprague Dawley (SD) adult male rats, weighing approximately 300 g (purchased at Shanghai SLAC Experimental Animal Limited Liability Company).
1.2 Main reagent pGC fu-RNAi-NC-LV recombinant viral carriers, containing only the green fluorescent protein GFP (Ubi-GFP, 5 × 109 TU / mL) (purchased from Shanghai Genechem Chemical Technology Co., Ltd.), OCT embedding medium (Leica, Germany).
1.3 Main instrument Rats 45 ° auricular stereotaxic apparatus (Steolting, USA), probe type in vivo fluorescence microscope (MKT, France), freezing microtome (Leica, Germany) and fluorescence microscope (Olympus, Japan).
1.4 Rat cerebral cortex micro-injections Rats were anaesthetized by intraperitoneal injection of 7% chloral hydrate (0.6 mL/100 g), and then fixed to a stereotaxic apparatus. After shaving with iodine and 75% alcohol for disinfection, a 1 cm long vertical incision at the top, with a cotton swab dipped in hydrogen peroxide to oxidise the periosteum, the front and back of the skull is exposed and marks are made. According to the Paxinos-Watson atlas [6], behind the anterior fontanelle 3.8 mm, side open 2.3 mm, depth 1.8 mm, to micro-inject the rat brain cortex location. Drill a vertical 0.5 mm diameter circular hole into the skull with an electric drill, use a micro-syringe to suck out the defined amount of Ubi-GFP, fix it on the designated location and insert it vertically by 1.8 mm until it reaches the parietal lobe and makes contact with the cortex (PtA) area, then inject the entire amount of the viral liquid by increments of 2 μL/10 min, the needle retention time being longer than 10 min. Slow injection from the syringe needle, suturing of incision and disinfection, intramuscular injection of penicillin to prevent infection. Conventional feeding of rats, followed by fluorescence observation 7 days later.
1.5 In vivo fluorescence microscopy imaging technique to observe rat cerebral cortex neurons 7% chloral hydrate (0.6 mL/100 g) is used and rats are anaesthetized by intraperitoneal injection, then shaven, then fixed in a stereotaxic apparatus. Early-phase exposure of skull hole and activation of in vivo fluorescence microscopy imaging recording system then takes place. Ajdustment of the in vivo fluorescence microscope diameter to 650 μm Proflex flexible fibre and insertion vertically into the rat's cerebral cortex, penetrating the meninges by 1.8 mm up to the PtA area's micro injection location, through the 488 nm laser activation, the acquisition signal band at 500 ~650 nm, the acquisition frequency of 12 / s is recorded in the in vivo fluorescence image (Fig. 1).
1.6 Frozen rat cerebral cortex section observation Excessive use of chloral hydrate aldehyde for intraperitoneal injections causes rats to die. After 4% poly-formaldehyde perfusion is fixed, remove micro injection area's brain tissue and commence gradient sucrose solution treatment.

2 Results
2.1 Rat neurons in vivo fluorescence microscopy imaging
Through the in vivo fluorescence imaging system, it is possible to acquire the target position's fluorescent images of the fibre-optic probe in real time. Furthermore, it is also possible to carry out micro-injections to the target location in the cerebral cortex of the rat, fine-tuning of the optical fibre probe, selection of two fields of vision to carry out the observation as well as recording within of the GFP tagged neurons' fluorescence signal dynamic changes. Using IC-Viewer software, the recorded data can be exported by means of image or picture formats. Fig. 2A and B show 6 consecutive screenshots from a period of 10 s, derived from separately collected two-stage data. In the diagram, the shapes of multiple tagged neurons can be observed at the corresponding point in time, the neuron soma's GFP fluorescence signal is quite clear. No obvious changes were observed in the GFP-tagged neuron during the recording time.

2.2 Observation of frozen cerebral cortex tissue
sections of rat
In order to verify the GFP signals verified by means of the in vivo fluorescence microscope, comparison with the same injected area of the frozen slices of the rat's cerebral cortex tissue is made. The fluorescence microscope observation shows the presence of GFP tags in neurons of the cerebral cortex (Fig. 3).

3 Discussion
Recombinant viral vectors containing GFP micro injection to a specific location of the animal's cerebral cortex and carrying out transfection to the micro-injected region's neurons can stably express GFP thereby facilitating the morphological observation of the target neurons [7]. During this experiment, after the micro-injection of Ubi-GFP into the cerebral cortex area, after 7 days of transfection, GFP neurons in the target area displayed a definite expression. We refer to parts tagged with GFP neurons as observed objects, in carrying out the in vivo fluorescence observation.

Fig. 4. Microphotograph showing the entire projecting view of imaging microprobe that is composed of around thirty thousand optical minifibers.

Probe-type in vivo fluorescence microscopy imaging technology is a hybrid of the two technologies of minute fibre optic probes and laser scanning technologies [8] in order to achieve real-time tracing recording of animals' internal signal trend change process features. Moreover, since the probe is only 650 μm in diameter and is made of softer material, it can satisfy the requirements for minimally invasive surgery on animals. The probe is composed of nearly 30,000 flexible fibres (Fig. 4 ), and this observation scope reaches 600 nm x 500 nm, with an axial resolution of 15 μm and a lateral resolution of 5 μm. It has a high sensitivity and can therefore be used for the in vivo tracing records of animal neuron signals [9]. In vivo fluorescence microscopy imaging is carried out with 488 nm laser activation, acquiring signal band of 400~650 nm, acquiring frequency of 12 width / s recording in vivo fluorescence imaging. The signal encoding equipment acquisition is 13 bits and the software can export multiple formats such as png, bmp and jpeg, etc., multiple format pictures as well as mpeg, mhd (raw format), etc. format images.

Through in vivo fluorescence microscopic imaging technology, we can observe the morphology of the rat cerebral cortex GFP-tagged neurons in real time.
Consecutive screenshots within 10 s showed that, although physiological factors such as the live animals' breathing and blood vessel pulsation produced slight impacts on the stability of the images, the interception of the target neurons in a short period of time still produced relatively stable results, the fluorescence signals of neuronal somas are relatively clear, and it is very consistent with the related literature [5]. This also suggests that the light during the recording process does not produce significant toxicity. The same frozen sections of the rat's cerebral cortex tissue were observed under a fluorescence microscope, the GFP-tagged neurons were observed, and then the in vivo fluorescence microscopy imaging techniques were further confirmed and the objectives were accomplished effectively. The above results show that the probe-type in vivo fluorescence microscope can stably record the neuronal morphology within a certain field of vision over a specific period of time and because of this it can be combined with stereotaxic techniques for the study of neuronal development and neuron migration observation methods.
Probe-type in vivo fluorescence imaging technology, because of its real-time acquisition of the special features of image data, combined with image analysis software, in addition to making the real-time observation of neuronal morphology possible, is also capable of acquiring and analysing data at high speeds during the fast-reaction phenomena of the body, such as calcium ion flow changes [5]. Apart from the neurons, it can also be used to carry out analysis of other tissue structures such as blood vessels, and can even carry out in vivo physiological synchronous information acquisition. Supporting the use of the the corresponding incision accessories, data records can be completed while the animal is awake. In addition, in view of the optical fibre probe activation wavelength of 488 nm, in optogenetics research, it can also be used for precise position activation of the ChR2 photosensitive protein. In summary, the introduction of optic-fibre in vivo fluorescence microscopy imaging technology has provided our technology platform's neuroscience experiments with more space to develop.

1. Langchuang’s Unique Characteristics
 Langchuang is a professional enterprise focused on corporate and business English training.
 Langchuang caters all its training courses to suit clients’ requirements and optimize their progress.
 Langchuang places strong focus on the effective execution of its methodology.
 Langchuang emphasizes improving the practical linguistic competence of its clients.

2. Training Outline

3. Training Process

4. Regulations of Training Process

6. Performance Evaluation
In order to obtain the best results, we carry out a 3-faceted evaluation process.
I. Trainee Performance: Our company generally employs two methods in assessing trainees’ results obtained through training:
a) TOEIC assessment through test questions which is already being used as a tool on a large scale by a wide range of foreign-owned corporations in order to assess the English proficiency levels of their respective employees (suitable for trainees with a good foundation in English).
b) Two separate tests set by our company in order to assess trainees’ test performance and training progress.
II. Trainer assessment (detailed assessment conducted by trainer)
III. Project consultant follow-up
We are committed to following the trainer and trainee assessment system as stipulated in the contract and if we fail to meet this target, we will make adjustments in order to maintain client satisfaction.

7. Training Recommendations
I. Initial Summary
In the past few years we have been fortunate to have had the opportunity to conduct your company’s English language training and thus, through several years of pleasant cooperation, we have become acquainted with most of your company’s staff and have noticed that the reason for less than ideal results in the cases of some students can be attributed to the some of the following factors:
a) Lack of confidence, shyness and fear of speaking;
b) Limited vocabulary;
c) Lack of grammatical proficiency
d) Upon reaching the final stages of the training courses, students sometimes begin to feel somewhat fatigued and thus their enthusiasm decreases.

II.Recommendations for Improvement

A. For the purposes of the upcoming course, we have adopted a new system of “speech duty” by means of which, in each lesson, 3 to 5 minutes is spent on listening to short speeches given by students in order to raise their confidence levels in spoken English. The content for these speech assignments is as follows:
Initial phase of the course: Self-selected topics, e.g.: one’s own life, favorite films, description of work day, etc.
Mid- and end-term phases: Topics selected by trainer with emphasis on working environment.

B. During each lesson, the trainer will have the following requirements from the trainees:
a) Vocabulary spelling listening exercise: Each lesson will require trainees to learn 10 to 15 new vocabulary items as this is a fundamental part of improving the trainee’s foundation in English;
b) Mastering definite sentence patterns or grammatical points through usage;
c) Mastering of 2 definite phrases or common usage items such as idioms, etc. in order to widen the trainees’ scope of knowledge in English usage.

C. Sustained raising of trainees’ interest
a) The trainer will focus on creating an atmosphere in order to enable the trainees to be able to better practice and will use roll-playing activities and exercises in conducting lessons;

b) The trainer will frequently divide the trainees into groups and encourage them to compete in specially-designed exercises in order to improve their skills through teamwork as well as increase their enthusiasm;
c) During the training sessions, the teacher may prepare certain multimedia materials and circumstantial simulations on conversations in the business environment or short foreign films for discussion in class.
D. Consolidation of Knowledge Outside the Classroom
a) After each class homework assignments will be given in order to consolidate the trainees’ knowledge of the material covered during the lessons.
b) There will be an average of 1 email exchanged between students and teachers every 4 lessons.
c) It will be recommended to trainees that they should establish practice groups as far as possible within their respective departments in order to benefit from after-class practice.

8. Scheduling and Grade Assessment
1. Training method: Group classes
2. Training frequency: a) Twice a week, two hours each time
b) Communication through email once a week
3. Training location: The company’s training room
4. Class grading: Trainees will be divided into three classes according to their respective proficiency levels: Beginner, intermediate and advanced levels.
5. Course structure: 60% teaching material content and 40% spent on work-related material
a) 60% Teaching Material Content:
Before the commencement of the course, we will conduct an exhaustive assessment on trainees’ vocabulary, grammar, pronunciation, fluency and written, etc. skills. Upon completion of the assessment, we will evaluate and consider all aspects of trainees’ abilities and respective proficiency levels as well as the HR department’s requirements and decide upon a suitable teaching syllabus text book for the purpose of conducting the course.
b) 40% Work-Related Topics
 During the training our trainer will collect and prepare a large amount of materials related to business English and gradually impart this material to the trainees, thus familiarizing them with its usage and enable them to utilize it immediately in their working environments whilst simultaneously building up their confidence in so doing.
 Secondly, the trainer will gather information regarding the trainees’ requests regarding their respective departments and positions and will address these gradually during the lessons. For example: the HR department’s correct form of written correspondence, the most suitable vocabulary used for the financial department’s reports, the technology department’s field-specific terminology, etc. in order to enable the staff members of different departments to learn and master the English skills necessary for their own respective responsibilities at work.

9. Follow-up
1. After completion of the assessment, the trainer will submit the teaching syllabus for the course to be taught and thus allow the company to better understand the scope and related issues of the course.
2. After the actual commencement of the course, a consultant will send a lesson plan of each lesson to the trainees as well as the HR department in order to enable trainees and to understand and prepare for each lesson in advance.
3. Every month, the course consultant will send a written report to the HR department. The report will include information relating to the course progression, student attendance records as well as homework completion, etc. in order to allow the HR department to conveniently have access to course progression information at any time.
4. The course consultant will audit classes at least 8 times during the course in order to supervise the trainer’s teaching methods as well as gather feedback from the trainees in order to be able to solve potential problems as soon as they may arise.
5. In the case of students whose absence rate is above normal or who find their studies to be exceedingly difficult, the course consultant will immediately inform the HR department of this by email in order to mutually decide upon the best solution for such cases.
6. During the mid-term phase of the course, there will be a mid-term assessment test. The test will include an oral section, a section to test listening ability as well as a general ability section. We will submit a written mid-term report to the HR department of your company.
7. During the graduation ceremony, certificates with summaries on student performance, etc. will be issued.
8. At the end of the course, we will submit written student records as part of our feedback to your company. The records will include information regarding student attendance, homework completion, behavior during class, performance during the training course as well as the end-term assessment and, in addition, will include recommendations for future courses.

10. Trainer Allocation
1. Before the commencement of the training, the company’s HR department and/or manager(s) in charge of training can make recommendations regarding your company’s trainer-related requirements. In addition, before the commencement of the course we will provide a free 2-hour consultation meeting in which teacher- and student-related issues as well as company expectations can be discussed. During this consultation meeting trainees will also be able to ask the trainer questions regarding the details of the course to be conducted.
2. It is recommended that students at the beginner and intermediate levels are taught by a native Chinese trainer as students at this level of proficiency are not yet advanced enough to keep up under the tuition of foreign trainers and native Chinese trainers are more apt to understand the difficulties commonly encountered by Chinese students and thus ensure faster progress.
3. It is recommended that students in the advanced level are taught by a foreign expert trainer as students at this level already have a definite foundation in English and require more than simple textual and conversational coaching and furthermore would benefit more from learning English through the medium of English.

I am a highly-qualified international translator and have been certified for proficiency at court-level interpreting and translation in seven different languages: i.e. English, Chinese (Mandarin), German, French, Spanish (Castilian standard), Japanese and Afrikaans. I am also able to translate from Portuguese and Dutch to English.

I have 3 and a half years experience in court interpreting and 11 years experience as a text translator in the above-mentioned languages.

Please feel free to contact me for a quotation on your translation project.

Looking forward to your correspondence and our cooperation.

Sincerely,

Don Hartig

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